Neuroinflammation can be monitored using fluorine-19 (19F)-containing nanoparticles and 19F MRI. Previously we studied neuroinflammation in experimental autoimmune encephalomyelitis (EAE) using room temperature (RT) 19F radiofrequency (RF) coils and low spatial resolution 19F MRI to overcome constraints in signal-to-noise ratio (SNR). This yielded an approximate localization of inflammatory lesions. Here we used a new 19F transceive cryogenic quadrature RF probe (19 F-CRP) that provides the SNR necessary to acquire superior spatially-resolved 19F MRI. First we characterized the signal-transmission profile of the 19 F-CRP. The 19 F-CRP was then benchmarked against a RT 19F/1H RF coil. For SNR comparison we used reference compounds including 19F-nanoparticles and ex vivo brains from EAE mice administered with 19F-nanoparticles. The transmit/receive profile of the 19 F-CRP diminished with increasing distance from the surface. This was counterbalanced by a substantial SNR gain compared to the RT coil. Intraparenchymal inflammation in the ex vivo EAE brains was more sharply defined when using 150 μm isotropic resolution with the 19 F-CRP, and reflected the known distribution of EAE histopathology. At this spatial resolution, most 19F signals were undetectable using the RT coil. The 19 F-CRP is a valuable tool that will allow us to study neuroinflammation with greater detail in future in vivo studies.

The auxiliary ␤-subunit KCNMB2 (␤ 2 ) endows the noninactivating large conductance Ca 2؉ -and voltagedependent potassium (BK) channel with fast inactivation. This process is mediated by the N terminus of KCNMB2 and closely resembles the "ball-and-chain"-type inactivation observed in voltage-gated potassium channels. Here we investigated the solution structure and function of the KCNMB2 N terminus (amino acids 1-45, BK␤ 2 N) using NMR spectroscopy and patch clamp recordings. BK␤ 2 N completely inactivated BK channels when applied to the cytoplasmic side; its interaction with the BK ␣-subunit is characterized by a particularly slow dissociation rate and an affinity in the upper nanomolar range. The BK␤ 2 N structure comprises two domains connected by a flexible linker: the pore-blocking "ball domain" (formed by residues 1-17) and the "chain domain" (between residues 20 -45) linking it to the membrane segment of KCNMB2. The ball domain is made up of a flexible N terminus anchored at a well ordered loop-helix motif. The chain domain consists of a 4-turn helix with an unfolded linker at its C terminus. These structural properties explain the functional characteristics of BK␤ 2 N-mediated inactivation.Large conductance K ϩ channels (BK 1 or MaxiK channels) are key modulators of excitability in many types of cell (1, 2).They are formed from four identical ␣-subunits encoded by the Slo gene and are activated by membrane depolarization and/or increase in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i (3-8)). This dual activation is unique among the large family of K ϩ channels and provides a direct feedback mechanism to regulate Ca 2ϩ influx. In many tissues, the activation gating of BK channels is modulated by accessory ␤-subunits, a family of membrane proteins (KCNMB) closely associated with the ␣-subunit (7). Four KCNMB proteins have been identified (KCNMB1-4), and they all share a prototypic topology of two transmembrane domains with intracellular N and C termini (9 -13). Functionally, each of these KCNMB proteins distinctly changes the rates of channel activation and deactivation as well as the apparent sensitivity of the channel for Ca 2ϩ (9). In addition, one of the ␤-subunits, KCNMB2 (␤ 2 ), was found to confer rapid and complete inactivation to the BK channel complex (11, 12) in a manner similar to that observed in chromaffin cells of the adrenal gland or in hippocampal CA1 neurons (14, 15). Analysis of this KCNMB2-mediated inactivation gating showed that it closely resembled the famous ball-andchain-type inactivation of voltage-gated K ϩ channels (Kv): (i) it is determined by the N terminus of KCNMB2; (ii) it occludes the open channel pore and competes with the pore-blocking agent tetraethylammonium (11, 12); (iii) recovery from inactivation is speeded up by an increase of the extracellular K ϩ concentration (11).Moreover, the N-terminal stretch of the KCNMB2 N terminus (19 amino acids) was shown to be a functional entity, i.e. its fusion to the N terminus of KCNMB1 (␤ 1 ) conferred rapid inactivation to this ...

Small conductance Ca2؉ -activated potassium (SK) channels underlie the afterhyperpolarization that follows the action potential in many types of central neurons. SK channels are voltage-independent and gated solely by intracellular Ca 2؉ in the submicromolar range. This high affinity for Ca 2؉ results from Ca 2؉ -independent association of the SK ␣-subunit with calmodulin (CaM), a property unique among the large family of potassium channels. Here we report the solution structure of the calmodulin binding domain (CaMBD, residues 396 -487 in rat SK2) of SK channels using NMR spectroscopy. The CaMBD exhibits a helical region between residues 423-437, whereas the rest of the molecule lacks stable overall folding. Disruption of the helical domain abolishes constitutive association of CaMBD with Ca 2؉ -free CaM, and results in SK channels that are no longer gated by Ca 2؉ . The results show that the Ca 2؉ -independent CaM-CaMBD interaction, which is crucial for channel function, is at least in part determined by a region different in sequence and structure from other CaMinteracting proteins.

Cumulative inactivation of voltage-gated (Kv) K؉ channels shapes the presynaptic action potential and determines timing and strength of synaptic transmission. Kv1.4 channels exhibit rapid "ball-and-chain"-type inactivation gating. Different from all other Kv␣ subunits, Kv1.4 harbors two inactivation domains at its N terminus. Here we report the solution structure and function of this "tandem inactivation domain" using NMR spectroscopy and patch clamp recordings. Inactivation domain 1 (ID1, residues 1-38) consists of a flexible N terminus anchored at a 5-turn helix, whereas ID2 (residues 40 -50) is a 2.5-turn helix made up of small hydrophobic amino acids. Functional analysis suggests that only ID1 may work as a pore-occluding ball domain, whereas ID2 most likely acts as a "docking domain" that attaches ID1 to the cytoplasmic face of the channel. Deletion of ID2 slows inactivation considerably and largely impairs cumulative inactivation. Together, the concerted action of ID1 and ID2 may promote rapid inactivation of Kv1.4 that is crucial for the channel function in short term plasticity.

Rapid N-type inactivation of voltage-dependent potassium (Kv) channels controls membrane excitability and signal propagation in central neurons and is mediated by protein domains (inactivation gates) occluding the open channel pore from the cytoplasmic side. Inactivation domains (ID) are donated either by the pore-forming ␣-subunit or certain auxiliary ␤-subunits. Upon coexpression, Kv␤1.1 was found to endow non-inactivating members of the Kv1␣ family with fast inactivation via its unique N terminus. Here we investigated structure and functional properties of the Kv␤1.1 N terminus (amino acids 1-62, ␤N-(1-62)) using NMR spectroscopy and patch clamp recordings. ␤N-(1-62) showed all hallmarks of N-type inactivation: it inactivated non-inactivating Kv1.1 channels when applied to the cytoplasmic side as a synthetic peptide, and its interaction with the ␣-subunit was competed with tetraethylammonium and displayed an affinity in the lower micromolar range. In aequous and physiological salt solution, ␤N-(1-62) showed no well defined three-dimensional structure, it rather existed in a fast equilibrium of multiple weakly structured states. These structural and functional properties of ␤N-(1-62) closely resemble those of the "unstructured" ID from Shaker B, but differ markedly from those of the compactly folded ID of the Kv3.4 ␣-subunit.Fast N-type inactivation of voltage-gated potassium (Kv) 1 channels shapes the action potential, governs the firing rate (spiking), and controls signal propagation in central neurons (1). Biophysically, N-type inactivation has long served as the model for gating transitions in ion channels and is realized by a "ball plug-in" mechanism. In this mechanism a protein domain termed "inactivation gate" or "inactivation ball" binds to its receptor at the inner vestibule of the open channel and thereby occludes the ion pathway (2-5). Such inactivation gates have been localized in the N terminus of various Kv␣ subunits and were shown to be functional entities, i.e. they conferred rapid inactivation to "ball-less" Kv␣ subunits when applied to the cytoplasmic side of the channels as synthetic peptides (5-8). Identical to protein-harbored inactivation domains, the synthetic gates interacted with channels in the open state, blocked the pore with low voltage dependence, and were competed with the channel blocker tetraethylammonium (TEA) (9 -11). Recently, the structures of the inactivation domains (ID) from Kv1.4 and Kv3.4 were determined with NMR spectroscopy. Both IDs were found to exhibit well defined and compact folding in aequous solution (12). In contrast, the ID from Shaker B showed no unique, folded structure (13,14).Besides with Kv␣ subunits owning an N-terminal ID, fast inactivation was observed for a subset of non-inactivating Kv␣1 channels when coexpressed with certain ␤-subunits (15-17). These auxiliary subunits constitute a family of cytoplasmic proteins (subdivided into subfamilies Kv␤1, -2, and -3) that are made up of two distinct regions: a highly conserved core region that shows homolo...